Method, instruments, and kit for autologous transplantation

ABSTRACT

An implantable article for cartilage repair by implantation in an animal. The implantable article includes a support matrix, and chondrocyte cells and a bio-compatible adhesive adhered to an edge of the support matrix, wherein the support matrix is absorbable by the animal. The support matrix is a biocompatible material such as collagen, and the adhesive is autologous fibrin.

STATEMENT OF RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/970,323, filed Oct. 3, 2001 (now U.S. Pat. No. 6,569,172), which is acontinuation-in-part of U.S. patent application Ser. No. 09/690,252,filed Oct. 17, 2000, (now U.S. Pat. No. 6,379,367), which is acontinuation of U.S. patent application Ser. No. 09/320,246, filed May26, 1999 (now U.S. Pat. No. 6,283,980), which in turn is a continuationof U.S. patent application Ser. No. 08/857,090, filed May 15, 1997 (nowU.S. Pat. No. 5,989,269), which is a continuation-in-part of U.S. patentapplication Ser. No. 08/704,891, filed Aug. 30, 1996 (now U.S. Pat. No.5,759,190), all of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The instant invention concerns the field of chondrocyte transplantation,bone and cartilage grafting, healing, joint repair and the prevention ofarthritic pathologies. In particular methods for the preparation of thegraft site, instruments for such preparation and for the autologoustransplantation of cells to the prepared graft site.

BACKGROUND OF THE INVENTION

More than 500,000 arthroplastic procedures and total joint replacementsare performed each year in the United States. Approximately the samenumber of similar procedures are performed in Europe. Included in thesenumbers are about 90,000 total-knee replacements and around 50,000procedures to repair defects in the knee per year in Europe. The numberof procedures are essentially the same in the U.S. (In: Praemer A.,Furner S., Rice, D. P., Musculoskeletal conditions in the United States,American Academy of Orthopaedic Surgeons, Park Ridge, Ill., 1992, 125).A method for regeneration-treatment of cartilage would be most useful,and could be performed at an earlier stage of joint damage, thusreducing the number of patients needing artificial joint replacementsurgery. With such preventative methods of treatment, the number ofpatients developing osteoarthritis would also decrease.

Techniques used for resurfacing the cartilage structure in joints havemainly attempted to induce the repair of cartilage using subchondraldrilling, abrasion and other methods whereby there is excision ofdiseased cartilage and subchondral bone, leaving vascularized cancellousbone exposed (Insall, J., Clin. Orthop. 1974,101,61; Ficat R. P. et al,Clin Orthop. 1979, 144, 74; Johnson L. L., In: Operative Arthroscopy,McGinty J. B., Ed., Raven Press, New York, 1991, 341).

Coon and Cahn (Science 1966, 153, 1116) described a technique for thecultivation of cartilage synthesizing cells from chick embryo somites.Later Cahn and Lasher (PNAS USA 1967, 58, 1131) used the system foranalysis of the involvement of DNA synthesis as a prerequisite forcartilage differentiation. Chondrocytes respond to both EFG and FGF bygrowth (Gospodarowicz and Mescher, J. Cell Physiology 1977, 93, 117),but ultimately lose their differentiated function (Benya et al., Cell1978, 15, 1313). Methods for growing chondrocytes were described and areprincipally being used with minor adjustments by Brittberg, M. et al.(New Engl. J. Med. 1994, 331, 889). Cells grown using these methods wereused as autologous transplants into knee joints of patients.Additionally, Kolettas et al. (J. Cell Science 1995, 108, 1991) examinedthe expression of cartilage-specific molecules such as collagens andproteoglycans under prolonged cell culturing. They found that despitemorphological changes during culturing in monolayer cultures (Aulthouse,A. et al., In Vitro Cell Dev. Biol., 1989,25,659; Archer, C. et al., J.Cell Sci. 1990,97,361; Hanselmann, H. et al., J. Cell Sci. 1994,107,17;Bonaventure, J. et al., Exp. Cell Res. 1994,212,97), when compared tosuspension cultures grown over agarose gels, alginate beads or asspinner cultures (retaining a round cell morphology) tested by variousscientists did not change the chondrocyte—expressed markers such astypes II and IX collagens and the large aggregating proteoglycans,aggrecan, versican and link protein did not change (Kolettas, E. et al.,J. Cell Science 1995,108,1991).

The articular chondrocytes are specialized mesenchymal derived cellsfound exclusively in cartilage. Cartilage is an avascular tissue whosephysical properties depend on the extracellular matrix produced by thechondrocytes. During endochondral ossification chondrocytes undergo amaturation leading to cellular hypertrophy, characterized by the onsetof expression of type X collagen (Upholt, W. B. and Olsen, R. R., In:Cartilage Molecular Aspects (Hall, B & Newman, S, Eds.) CRC Boca Raton1991, 43; Reichenberger, E. et al., Dev. Biol. 1991, 148, 562; Kirsch,T. et al., Differentiation, 1992, 52, 89; Stephens, M. et al., J. CellSci. 1993, 103, 1111).

Excessive degradation of type II collagen in the outer layers orarticular surfaces of joints is also caused by osteoarthritis. Thecollagen network is accordingly weakened and subsequently developsfibrillation whereby matrix substances such as proteoglycans are lostand eventually displaced entirely. Such fibrillation of weakenedosteoarthritic cartilage can reach down to the calcified cartilage andinto the subchondral bone (Kempson, G. E. et al., Biochim. Biophys. Acta1976, 428, 741; Roth, V. and Mow, V. C., J. Bone Joint Surgery, 1980,62A, 1102; Woo, S. L.-Y. et al., in Handbook of Bioengineering (R.Skalak and S. Chien eds.), McGraw-Hill, New York, 1987, pp. 4.1–4.44).

Descriptions of the basic development, histological and microscopicanatomy of bone, cartilage and other such connective tissues can befound for example in Wheater, Burkitt and Daniels, Functional Histology,2nd Edition, (Churchill Livingstone, London, 1987, Chp. 4). Descriptionsof the basic histological anatomy of defects in bone, cartilage andother connective tissue can be found for example in Wheater, Burkitt,Stevens and Lowe, Basic Histopathology (Churchill Livingstone, London,1985, Chp. 21).

Despite the advances in cultivating chondrocytes, and manipulating boneand cartilage, there has not been great success with the attempts totransplant cartilage or chondrocytes for the repair of damagedarticulating surfaces. The teachings of the instant invention providefor effective, and efficient means of promoting the transplantation ofcartilage and/or chondrocytes into a defect in an articulating joint orother cartilage covered bone surface, whereby cartilage is regeneratedto fix the defect. The instant invention also provides for surgicalinstruments which are designed to prepare the graft site so as tofacilitate the efficient integration of grafted material to the graftsite.

BRIEF SUMMARY OF THE INVENTION

The instant invention provides a method for the effective treatment ofarticulating joint surface cartilage by the transplantation ofchondrocytes in a suitable matrix, to a surface to be treated, with ahemostatic barrier and a cell-free covering-patch comprising; firstplacing a hemostatic barrier proximal to the surface to be treated,placing chondrocytes in a suitable matrix upon the surface to be treateddistal to the hemostatic barrier, covering the surface to be treatedwith a cell-free covering-patch. A hemostatic barrier, as will befurther described below, is a barrier which inhibits the penetration ofvascularizing cells and tissue into the grafted material. In particular,the instant method provides for a hemostatic barrier that is aresorbable, semi-permeable material which inhibits or prohibits vascularinfiltration through the barrier. In one embodiment the hemostaticbarrier contains collagen. Cell-free, is used herein as in the art, andmeans a material that is substantially free from intact cells which arecapable of further cell division, promulgation or biological activity.In a preferred embodiment, a cell-free material is free from all intactnucleated cells. In one embodiment, the instant method encompasses theuse of a cell-free covering patch which contains a semi-permeablecollagen matrix. In one preferred embodiment of the method, the poroussurface of the cell-free covering-patch is directed towards the implantmaterial.

The instant invention further provides for the autologoustransplantation of collagen or chondrocytes to a graft site, wherein thegraft site has first been prepared by surgical manipulation to betteraccept the grafted material. In one embodiment, the graft site issculpted such that the walls of the graft site are contoured in anundulating pattern such that the grafted material, when placed withinthe graft site and expanded to contact the graft site wall, providesresistance against removal or expulsion of the entire graft from thegraft site. The instant invention further provides for surgicalinstruments designed to sculpt the graft site as taught by the method ofthe invention.

The invention further provides for a kit for cartilage and/orchondrocyte transplantation onto the surface of an articular jointwherein said kit comprises a hemostatic barrier, cell-freesemi-permeable covering-patch, and organic glue. In a furtherembodiment, the kit can optionally further provide one or more surgicalinstruments which can be used to sculpt the graft site in accordancewith the methods of the instant invention.

The present invention further provides an implant for cartilage repairin an animal. In one embodiment, the implant is 1) a support matrix, 2)chondrocyte cells, and 3) a bio-compatible adhesive adhered to an edgeof the support matrix. In one embodiment, the support is absorbable bythe animal.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood by examining thefollowing figures which illustrate certain properties of the instantinvention wherein:

FIG. 1A is a drawing showing a typical articulating end of a bone.Typically, the bone material is covered on the articulating surface witha cartilaginous cap (shown by cross-hatching).

FIG. 1B shows an example of where a defect or injury to thecartilaginous cap occurs (gap in the cross-hatching), and such a defectcan be treated directly, enlarged slightly, or sculpted to accept thegrafted material by surgical procedures prior to treatment.

FIG. 1C shows how the hemostatic barrier (solid black, numbered 1) isplaced within the defect in the cartilage cap to inhibit or preventvascularization into regenerating cartilage, from the underlying bone.The chondrocytes to be implanted into the defect cavity are then layeredon top of the hemostatic barrier.

FIG. 2 is a drawing showing the treated defect (gap in cross-hatchedarea) in the cartilaginous cap (cross-hatched area) covered by acell-free semi-permeable material (solid black, numbered 2) which isused to form a cap/patch or bandage over the defect site. This cap isfixed in place, either sutured to the edge of the cavity into healthycartilage, or otherwise attached. This cap is covering the defectivearea of the joint into which the glue and culturedchondrocytes/cartilage transplant has been placed.

FIG. 3A is a diagram illustrating the differential response tocompression and shearing forces by harder and softer cartilage withsubsequent zone of demarcation.

FIG. 3B illustrates the graft site, after the defect has been sculptedto have undulating walls.

FIG. 3C illustrates the sculpted graft site with the hemostatic barrier(1), transplanted material (3), and cell-free covering-patch (2) inplace within the articular surface cartilage (4).

FIG. 4A illustrates one embodiment of the surgical device of the instantinvention showing cutting teeth (5) and protruding placement pin (6).

FIG. 4B illustrates a second embodiment of the surgical device of theinstant invention.

FIG. 5 is a diagram illustrating the modified differential response tocompression and shearing forces by harder cartilage and softer cartilageafter sculpting the graft site.

FIG. 6A is an MRI image of a pig knee showing cartilage defect in left(medial) condyle.

FIG. 6B is an MRI image of the same pig knee three months aftertreatment.

FIG. 7 illustrates a graft site or defect (23) without a hemostaticbarrier, including a mixture of transplanted cells (21) andbio-compatible glue (22) on a support matrix (18) having a porous side,a cartilaginous material and transplanted material interface (30),cartilage (10), a bone material and cartilaginous material interface(12), a resorbable pin (24) and a bone material (14).

DETAILED DESCRIPTION OF THE INVENTION

This invention concerns the use of certain products that inhibit theformation of vascular tissue, for instance such as capillary loopsprojecting into the cartilage being established, during the process ofautologous transplantation of chondrocytes into defects in thecartilage. The formation of vascular tissue from the underlying bonewill tend to project into the new cartilage to be formed leading toappearance of cells other than the mesenchymal specialized chondrocytesdesired.

The contaminating cells introduced by the vascularization may give riseto encroachment and over-growth into the cartilage to be formed by theimplanted chondrocytes. One of the types of commercial products whichcan be used in this invention is Surgicel® (Ethicon Ltd., UK) which isabsorbable after a period of 7–14 days. The use of this material in themethod of the instant invention is contrary to the normal use of ahemostatic device, such as Surgicel® as it is described in the packageinsert from Ethicon Ltd.

Surprisingly, we have found that in a situation where you wish toinhibit re-vascularization into cartilage, a hemostatic material willact like a gel-like artificial coagulate. If red blood cells should bepresent within the full-thickness defect of articular cartilage that iscapped by such a hemostatic barrier, these blood cells will bechemically changed to hematin, and thus rendered unable to inducevascular growth. Thus, a hemostatic product used as a re-vascularizationinhibitory barrier with or without fibrin adhesives, such as for examplethe Surgicel®, is effective for the envisioned method as taught by theinstant invention. Another part of this invention is the use of acell-free component, that is used as a patch covering the defective areaof the joint into which the cultured chondrocytes/cartilage are beingtransplanted, using autologous chondrocytes for the transplantation. Themethod of the invention also contemplates the use of suitable allogenicchondrocytes or xenogenic chondrocytes for the repair of a cartilagedefect.

Thus the instant invention teaches methods for effective repair ortreatment of cartilage defects in articular joint bone surfaces whichcomprises administering an agent or device to block vascular invasioninto the cartilage site to be repaired, and also providing for acell-free barrier which will isolate the repair site and keeptransplanted cells in place. The instant invention also provides for akit comprising a hemostatic barrier component for insertion into thesite to be repaired, such that there is effective inhibition ofvascularization into the site to be repaired; and once the chondrocytesto be transplanted are placed into the site to be repaired, a cell-freesemi-permeable barrier is capped over the repair site such that thetransplanted chondrocytes are held in place, but are still able to gainaccess to nutrients.

Certain aspects of the invention have been exemplified using an in vitrosystem to study the behavior of the chondrocytes when in contact with acertain product or a combination of certain products that inhibit theformation of vascular tissue. This in vitro testing predicts the abilityof certain tested materials to inhibit vascularization, as will occur invivo where capillary loops project into the cartilage being establishedduring the process of autologous transplantation of chondrocytes intodefects in the cartilage.

Suitable hemostatic products will be characterized by having the abilityto inhibit the growth, or invasion of vascular tissue, osteocytes,fibroblasts etc. into the developing cartilage. A suitable hemostaticmaterial will achieve the goal of the method of the instant invention inthat vascular and cellular invasion into developing cartilage should beprevented in order to optimize the formation of cartilage and achieverepair of the full-thickness of any defects in the articular cartilage.Ideally, the hemostatic barrier will be stable for an extended period oftime sufficient to allow for full cartilage repair, and then be able tobe resorbed or otherwise broken down over time. One material identifiedas suitable is called Surgicel® W1912 (an absorbable hemostat containingoxidized regenerated sterile cellulose; Lot GG3DH, Ethicon Ltd. UK).Another example of a suitable material is BioGide® (a commerciallyavailable type I collagen matrix pad; Geistlich Sohne, Switzerland).

Suitable organic glue material can be found commercially, such as forexample Tisseel® or Tissucol® (fibrin based adhesive; Immuno AG,Austria), Adhesive Protein (Cat. #A-2707, Sigma Chemical, USA), and DowComing Medical Adhesive B (Cat. #895-3, Dow Corning, USA).

The surgical instruments contemplated by the instant invention can bemanufactured from metal and/or plastic suitable for making single-usedisposable, or multi-use reusable surgical instruments. The cuttinginstrument may contain cutting teeth that are fully circular or flat, oranything in between. As cartilage is a relatively soft material it maybe advantageous to manufacture hardened plastic cutting edges which willbe able to sculpt cartilage without being able to damage bone. Suchcutting instruments can be manufactured to incorporate openings foradministration of fluid, suction removal of cutting debris and fluid,and fiber optic threads for illumination and visualization of the defectsite.

In another embodiment, the present invention includes cells 21 and glue22 combined together as (1) a mixture of glue 22 and cells 21 or (2) oneor more alternating layers of cells 21 and glue 22 on support matrix 18.

In one such embodiment, it is contemplated that cells 21 are autologouschondrocyte cells to be transplanted into a defect 23. Chondrocytes 21are mixed, either homogeneously or non-homogeneously, with a suitableglue 22 before application of the chondrocyte/glue mixture to supportmatrix 18. Preferably, glue 22 and chondrocytes 21 are mixed immediately(that is, in the operating theater) before applying glue 22 and cells 21to support matrix 18, and implantation of the combination of glue 22,cells 21, and support matrix 18 into defect 23. Alternatively cells 21and glue 22 are alternately applied in one or more layers to supportmatrix 18. In one embodiment, glue 22 is a bio-compatible glue, such asa fibrin glue, and more specifically either an autologous fibrin glue ora non-autologous fibrin glue. Preferably, an autologous fibrin glue isused.

When chondrocytes 21 are combined with glue 22 on support matrix 23, theglue/chondrocyte/support matrix combination is implanted into defect 23either with or without a hemostatic barrier (as described herein)between bone 14 and the glue/chondrocyte/support matrix combination. Inone embodiment, as shown in FIG. 2, treated defect 23 does not contain ahemostatic barrier between bone 14 and the glue/chondrocyte/supportmatrix combination.

In one embodiment, shown in FIG. 7, the treatment of defect 23 accordingto the present invention similarly is accomplished without the use of ahemostatic barrier between the glue/chondrocyte/support matrixcombination and the base of defect 23.

Additionally, in the embodiment shown in FIG. 7, the combination of glue22 and chrondrocytes 21 is present only on a portion of support matrix18. For example, glue 22 and chondrocytes 21 preferably are only on anouter edge of a porous side of support matrix 18. Alternatively,chondrocytes 21 and/or glue 22 penetrate into a porous side of supportmatrix 18 to a desired level, for example 2 to 50% of the depth ofsupport matrix 18. Typically the porosity of support matrix 18 isgreatest nearest a rough side, i.e., the side on which cells 21 and glue22 are deposited, and least porous on the opposite side of supportmatrix 18. As further illustrated by FIG. 7, the porosity of supportmatrix 18 is indicated by shading, where the darker shading indicatesless porosity, and the lighter shading indicates greater porosity.

In yet another embodiment, chondrocytes 21 are applied to support matrix18, then covered with a layer of a suitable bio-compatible glue 22.Although a single layer of chondrocytes 21 on support matrix 18 and asingle layer of glue 22 positioned over chondrocytes 21 is sufficientfor effective treatment of a defect, more than one alternating layer ofglue 22 and chondrocytes 21 can be used. Alternatively, a layer ofbio-compatible glue 22 can be initially deposited on support matrix 18,followed by a layer of chondrocytes 21 deposited on glue 22.

In one embodiment, support matrix 18 is a sheet-like member capable ofsupporting growth of chondrocytes 21 and of providing physical integrityto the implantable article to facilitate manipulation thereof. In anembodiment, support matrix 18 includes polypeptides or proteins such ascollagen. In particular, support matrix 18 includes equine, porcine,bovine, ovine and chicken collagen. Additionally, the collagen can beeither Type I or Type II collagen. Examples of appropriate collagensupport matrices 18 include ChondraGide® and BioGide® (commerciallyavailable type I collagen matrix pads; available from Geistlich Sohne,Switzerland). Additionally, support matrix 18 is reversibly deformableand has either a rough side that is porous, as described above, or asmooth side, or two rough sides. Although either side can be porous, inone embodiment as described herein, the rough side has greater porosityrelative to the smooth side. Support matrix 18 and cells 21 positionedthereon can be optionally held in place in the defect by additionalbio-compatible glue 22 and/or one or more biocompatible resorbable pins24, as shown in FIG. 7.

In one embodiment, one or more portions of support matrix 18 is eithersolid or gel-like in character.

Certain aspects of the instant invention may be better understood asillustrated by the following examples, which are meant by way ofillustration and not limitation.

EXAMPLE 1

In order for the Surgicel® to be used according to the invention forpreventing development of blood vessels into autologous implantedcartilage or chondrocytes, Surgicel® was first treated with a fixative,such as glutaric aldehyde. Briefly, Surgicel® was treated with 0.6%glutaric aldehyde for 1 minute, followed by several washings toeliminate glutaric aldehyde residues that may otherwise be toxic totissue. Alternatively, the Surgicel® was treated with the fibrinadhesive called Tisseel® prior to treatment with glutaric aldehyde asdescribed in Example 2. It was found that the Surgicel® fixated forinstance with a fixative such as glutaric aldehyde, washed with sterilephysiological saline (0.9%) and stored in refrigerator, does notdissolve for 1 to 2 months. Generally, Surgicel® is resorbed in a periodbetween 7 and 14 days. This time would be too short, because a longertime is needed in preventing the development of blood vessels orvascularization as such from the bone structure into the implantedcartilage before the implanted chondrocytes have grown into a solidcartilage layer getting its nutrition requirements from the neighboringcartilage. In other words sufficient inhibition of the vascularizationis needed for a longer time, such as for instance one month. Therefore,the product should not be absorbed significantly prior to that time. Onthe other hand resorption is needed eventually. Hence, the organicmaterial used as an inhibiting barrier shall have these capabilities,and it has been found that the Surgicel® treated in this manner providesthat function.

EXAMPLE 2

The Surgicel® was also coated with an organic glue, in this example theglue used was Tisseel® but others can also be used. This product,together with the Surgicel® produces a useable barrier for theparticular purpose of the invention. Any other hemostat or vascularinhibiting barrier could be used. The Tisseel® was mixed as describedbelow. The Surgicel® was then coated with Tisseel® by spraying theSurgicel® material on both sides until soaked. The Tisseel® (fibringlue) was then allowed to solidify at room temperature. Immediatelyprior to completed solidification, the coated Surgicel® was then placedin 0.6% glutaric aldehyde for 1 minute and then washed with sterilephysiological (0.9%) saline. The pH was then adjusted by PBS and/or withNaOH until pH was stable at 7.2 to 7.4. Afterwards the thus treatedSurgicel® was then washed in tissue culture medium such as minimumessential medium/F12 with 15 mM Hepes buffer.

As mentioned in this example we have used Tisseel® as the fibrinadhesive to coat the Surgicel®. Furthermore the fibrin adhesive or gluemay also be applied directly on the bottom of the lesion towards thebone, on which the Surgicel® is glued. The in vitro system used, in lieuof in vivo testing, consisted of a NUNCLON™ Delta 6-well steriledisposable plate for cell research work (NUNC. InterMed, Roskilde,Denmark). Each well measures approximately 4 cm in diameter.

In the invention the fibrin adhesive can be any adhesive which togetherwith the fibrin component will produce a glue that can be tolerated inhumans (Ihara, N, et al., Bums Inel. Therm. Inj., 1984, 10, 396). Theinvention also anticipates any other glue component that can be used inlieu of the fibrin adhesive. In this invention we used Tisseel® orTissucol® (Immuno AG, Vienna, Austria). The Tisseel® kit consists of thefollowing components:

-   -   Tisseel®, a lyophilized, virus-inactivated Sealer, containing        clottable protein, thereof: fibrinogen, Plasma fibronectin (CIG)        and Factor XIII, and Plasminogen;    -   Aprotinin Solution (bovine);    -   Thrombin 4 (bovine);    -   Thrombin 500 (bovine); and    -   Calcium Chloride solution.

The Tisseel® kit contains a DUPLOJECT® Application System. The fibrinadhesive or the two-component sealant using Tisseel® Kit is combined inthe following manner according to the Immuno AG product insert sheet.

EXAMPLE 3

Chondrocytes were grown in minimal essential culture medium containingHAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO₂incubator at 37° C. and handled in a Class 100 laboratory at VerigenEurope A/S, Symbion Science Park, Copenhagen, Denmark. Othercompositions of culture medium may be used for culturing thechondrocytes. The cells were trypsinized using trypsin EDTA for 5 to 10minutes and counted using Trypan Blue viability staining in aBurker-Turk chamber. The cell count was adjusted to 7.5×10⁵ cells perml. One NUNCLON™ plate was uncovered in the Class 100 laboratory.

The Surgicel® hemostatic barrier was cut to a suitable size fitting intothe bottom of the well in the NUNCLON™ tissue culture tray. In this casea circle, of a size of approximately 4 cm (but could be of any possiblesize) and placed under aseptic conditions on the bottom in well in aNUNCLON™ Delta 6-well sterile disposable plate for cell research work(NUNC, InterMed, Roskilde, Denmark). The hemostatic barrier to be placedon the bottom of the well was pre-treated as described in Example 1.This treatment delays the absorption of the Surgicel significantly. Thishemostatic barrier was then washed several times in distilled water andsubsequently several times until non-reacted glutaraldehyde was washedout. A small amount of the cell culture medium containing serum wasapplied to be absorbed into the hemostatic barrier and at the same timekeeping the hemostatic barrier wet at the bottom of the well.

Approximately 10⁶ cells in 1 ml culture medium were placed directly ontop of the hemostatic barrier, dispersed over the surface of thehemostatic barrier pre-treated with 0.4% glutaraldehyde as describedabove. The plate was then incubated in a CO₂ incubator at 37° C. for 60minutes. An amount of 2 to 5 ml of tissue culture medium containing 5 to7.5% serum was carefully added to the well containing the cells avoidingsplashing the cells by holding the pipette tip tangential to the side ofthe well when expelling the medium. It appeared that the pH of themedium was too low (pH˜6.8). The pH was then adjusted to 7.4 to 7.5. Thenext day some chondrocytes had started to grow on the hemostaticbarrier, arranged in clusters. Some of the cells had died due to the lowpH exposure prior to the adjustment of the pH. The plate was incubatedfor 3 to 7 days with medium change at day 3.

At the end of the incubation period the medium was decanted, and coldrefrigerated 2.5% glutaraldehyde containing 0.1M sodium salt ofdimethylarsinic acid, (also called sodium cacodylate, pH is adjustedwith HCl to 7.4), was added as a fixative for preparation of the celland supporter (hemostatic barrier) for later preparation for electronmicroscopy.

EXAMPLE 4

Chondrocytes were grown in minimal essential culture medium containingHAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO₂incubator at 37 C. and handled in a Class 100 laboratory at VerigenEurope A/S, Symbion Science Park, Copenhagen, Denmark. Othercompositions of culture medium may be used for culturing thechondrocytes. The cells were trypsinized using trypsin EDTA for 5 to 10minutes and counted using Trypan Blue viability staining in aBurker-Turk chamber. The cell count was adjusted to 7.5×10⁵ cells perml. One NUNCLON™ plate was uncovered in the Class 100 laboratory.

The Surgicel® (for use as the hemostatic barrier) was treated with 0.6%glutaric aldehyde for one minute as described in Example 1, and washedwith 0.9% sterile sodium chloride solution or, preferably, with a buffersuch as a PBS buffer or the culture medium such as MEM/F12, because thepH after the glutaric aldehyde treatment is 6.8 and should preferably be7.0 to 7.5. The Tisseel® was applied on both sides of the Surgicel®using the DUPLOJECT® system, thus coating both sides of the Surgicel®,the patch intended to be used, with fibrin adhesive. The glue is left todry under aseptic condition for at least 3 to 5 minutes. The “coated”hemostatic barrier was placed on the bottom of the well in a NUNCLON™Delta 6-well sterile disposable plate for cell research work. A smallamount of tissue culture medium containing serum was applied to beabsorbed into the hemostatic barrier. Approximately 10⁶ cells in 1 mltissue culture medium containing serum was placed directly on top of theHemostat, dispersed over the surface of the hemostatic barrier. Theplate was then incubated in a CO₂ incubator at 37° C. for 60 minutes. Anamount of 2 to 5 ml of tissue culture medium containing 5 to 7.5% serumwas carefully added to the well containing the cells avoiding splashingthe cells by holding the pipette tip tangential to the side of the wellwhen expelling the medium. After 3 to 6 days, microscopic examinationshowed that the cells were adhering to and growing into the Surgicel® ina satisfactory way suggesting that Surgicel® did not show toxicity tothe chondrocytes and that the chondrocytes grew in a satisfactory mannerinto the Surgicel®.

The plate was incubated for 3 to 7 days with medium change at day 3. Atthe end of the incubation period the medium was decanted, and coldrefrigerated, and 2.5% glutaraldehyde containing 0.1M sodium salt ofdimethylarsinic acid, also called sodium cacodylate, pH is adjusted withHCl to 7.4, was added as a fixative for preparation of the cell andsupporter (hemostatic barrier) for later preparation for electronmicroscopy.

EXAMPLE 5

Chondrocytes were grown in minimal essential culture medium containingHAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO₂incubator at 37 C. and handled in a Class 100 laboratory at VerigenEurope A/S, Symbion Science Park, Copenhagen, Denmark. The cells weretrypsinized using trypsin EDTA for 5 to 10 minutes and counted usingTrypan Blue viability staining in a Burker-Turk chamber. The cell countwas adjusted to 7.5×10⁵ to 2×10⁶ cells per ml. One NUNCLON™ plate wasuncovered in the Class 100 laboratory.

It has been found that the Bio-Gide® can be used as a resorbable bilayermembrane which will be used as the patch or bandage covering thedefective area of the joint into which the cultured chondrocytes arebeing transplanted as well as the hemostatic barrier. The Bio-Gide® is apure collagen membrane obtained by standardized, controlledmanufacturing processes (by E. D. Geistlich Sohne AG, CH-6110 Wolhusen).The collagen is extracted from veterinary certified pigs and iscarefully purified to avoid antigenic reactions, and sterilized indouble blisters by gamma irradiation. The bilayer membrane has a poroussurface and a dense surface. The membrane is made of collagen type I andtype III without further cross-linking or chemical treatment. Thecollagen is resorbed within 24 weeks. The membrane retains itsstructural integrity even when wet and it can be fixed by sutures ornails. The membrane may also be “glued” using fibrin adhesive such asTisseel® to the neighboring cartilage or tissue either instead ofsutures or together with sutures.

The Bio-Gide® was un-covered in a class 100 laboratory and placed underaseptic conditions on the bottom of the wells in a NUNCLON™ Delta 6-wellsterile disposable plate for cell research work,—either with the poroussurface of the bilayer membrane facing up or with the dense surfacefacing up. Approximately 10⁶ cells in 1 ml tissue culture mediumcontaining serum was placed directly on top of the Bio-Gide®, dispersedeither over the porous or the dense surface of the Bio-Gide). The platewas then incubated in a CO₂ incubator at 37° C. for 60 minutes. Anamount of 2 to 5 ml of tissue culture medium containing 5 to 7.5% serumwas carefully added to the well containing the cells avoiding splashingthe cells by holding the pipette tip tangential to the side of the wellwhen expelling the medium.

On day 2 after the chondrocytes were placed in the well containing theBio-Gide® the cells were examined in a Nikon Inverted microscope. It wasnoticed that some chondrocytes had adhered to the edge of the Bio-Gide.It was of course not possible to look through the Bio-Gide® itself usingthis microscope.

The plate was incubated for 3 to 7 days with medium change at day 3. Atthe end of the incubation period the medium was decanted, and coldrefrigerated 2.5% glutaraldehyde containing 0.1M sodium salt ofdimethylarsinic acid, also called sodium cacodylate, pH is adjusted withHCl to 7.4, was added as fixative for preparation of the cell and theBio-Gide® supporter with the cells either cultured on the porous surfaceor the dense surface. The Bio-Gide® patches were then sent for electronmicroscopy at Department of Pathology, Herlev Hospital, Denmark.

The electron microscopy showed that the chondrocytes cultured on thedense surface of the Bio-Gide® did not grow into the collagen structureof the Bio-Gide®, whereas the cells cultured on the porous surface didindeed grow into the collagen structure and furthermore, showed presenceof proteoglycans and no signs of fibroblast structures. This resultshows that when the collagen patch, as for instance a Bio-Gide® patch issewn as a patch covering a cartilage defect the porous surface shall befacing down towards the defect in which the cultured chondrocytes are tobe injected. They will then be able to penetrate the collagen andproduce a smooth cartilage surface in line with the intact surface, andin this area a smooth layer of proteoglycans will be built up. Whereas,if the dense surface of the collagen is facing down into the defect thechondrocytes to be implanted will not integrate with the collagen, andthe cells will not produce the same smooth surface as described above.

EXAMPLE 6

Chondrocytes were grown in minimal essential culture medium containingHAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO₂incubator at 37° C. and handled in a Class 100 laboratory at VerigenEurope A/S, Symbion Science Park, Copenhagen, Denmark. The cells weretrypsinized using trypsin EDTA for 5 to 10 minutes and counted usingTrypan Blue viability staining in a Burker-Turk chamber. The cell countwas adjusted to 7.5×10⁶ to 2×10⁶ cells per ml. One NUNCLON™ plate wasuncovered in the Class 100 laboratory.

The Bio-Gide® used as a resorbable bilayer membrane may also be usedtogether with an organic glue such as Tisseel® with additional,significantly higher content of Aprotinin than normally found inTisseel®, as described in the product insert. By increasing the contentof Aprotinin to about 25,000 KIU/ml, the resorption of the material willbe delayed by weeks instead of the normal span of days.

To test this feature in vitro, the Tisseel® is applied to the bottom ofthe well of the NUNCLON™ plate, and allowed to solidify incompletely. Acollagen patch such as a Bio-Gide® is then applied over the Tisseel® andglued to the bottom of the well. This combination of Bio-Gide® andTisseel® is designed to be a hemostatic barrier that will inhibit orprevent development or infiltration of blood vessels into thechondrocyte transplantation area. This hybrid collagen patch can now beused for both a hemostatic barrier at the bottom of the lesion (mostproximal to the surface to be repaired) as well as a support forcartilage formation because the distal surface can be the porous side ofthe collagen patch and thus encourage infiltration of chondrocytes andcartilage matrix. Thus, this hybrid collagen patch can also be used tocover the top of the implant with the collagen porous surface directeddown towards the implanted chondrocytes and the barrier forming the top.The hybrid collagen patch, with elevated Aprotinin component may also beused without any organic glue such as Tisseel® and placed within thedefect directly, adhering by natural forces. Thus the collagen patch canbe used both as the hemostatic barrier, and the cell-free covering ofthe repair/transplant site, with the porous surfaces of the patchesoriented towards the transplanted chondrocytes/cartilage. Anothervariant would use a collagen patch which consists of type II collagen(i.e. from Geistlich Sohne AG, CH-6110 Wolhusen).

Thus the instant invention provides for a hybrid collagen patch wheresaid patch is a collagen matrix with elevated levels of aprotinincomponent, preferably about 25,000 KIU/ml, in association with anorganic matrix glue, where the collagen component is similar to theBio-Gide® resorbable bilayer material or Type II collagen, and theorganic glue is similar to the Tisseel® material. In another embodiment,the hybrid collagen patch does not use any organic glue to adhere to thesite of the repair.

EXAMPLE 7

Because of the weakened structure of osteoarthritic cartilage, adherenceof cultured autologous chondrocytes transplanted to a graft site indefective cartilage may be inhibited, thus creating a marginal zone(zone of demarcation) between the newly implanted cartilage/chondrocytesand the surrounding established cartilage. This marginal zone will bemost pronounced if the graft site is prepared for the graft by creatingstraight, smooth walls cut in a linear fashion. The shearing andcompression forces across such a marginal zone (as illustrated in FIG.3A) will exert great force to dislodge the graft when the graft site iscut in a linear fashion. This marginal zone, and differential movementof materials along this zone will inhibit confluent healing between thegrafted material and the surrounding material. In many cases the graftmaterial is softer than the surrounding material, however, in someinstances of osteoarthritis disease, the surrounding cartilage may infact be softer than the implanted chondrocytes/cartilage.

Therefore, in order to solve this problem, the method of the inventionteaches the use of surgical instruments to sculpt the walls of the graftsite such that the walls are non-linear, and thus provide for undulatedsurfaces. It is also possible to shape the graft site such that thediameter of the site proximal to the bone surface is of a greaterdimension then the opening distal to the bone, and at the surface of thecartilage. However, the preferred embodiment describes the sculpting ofthe walls of the graft site in an fashion similar to a threaded openingfor receiving a bolt or screw (as illustrated in FIG. 3B), thusproviding mechanical resistance to the compression and or ejection ofthe grafted material from the graft site which can be described as“male” and “female” threading.

The surgical instruments contemplated by the instant invention can bemanufactured from metal and/or plastic suitable for making single-usedisposable, or multi-use reusable surgical instruments. As cartilage isa relatively soft material it may be advantageous to manufacturehardened plastic cutting edges which will be able to sculpt cartilagewithout being able to damage bone. Such cutting instruments can bemanufactured to incorporate openings for administration of fluid,suction removal of cutting debris and fluid, and fiber optic threads forillumination and visualization of the defect site. In certainembodiments of the instrument, the base of the instrument may haveprotruding point or pin-like structure which will assist in guiding andplacing the instrument in the graft site. Of course such a pin would bedesigned to minimized damage to the underlying bone.

While the cutting surface of the instrument may be single toothed, ormulti-toothed, or describe a screw-like pattern such as that in a metaltap used to generate threaded holes in metal parts, the characteristicrequired of the cutting instrument is that the resulting sculpted sidesof the graft site is undulated, and non-linear. For example, in certainembodiments, the cutting edge of the instrument can be shaped similar tothat shown in FIG. 4A, or as in FIG. 4B. The cutting edge maybe flat, orcircular in that it wraps around the diameter of the cutting instrument.Many other shapes can be designed to accomplish the purpose of themethod of the invention to create an interface which provides formechanical resistance to differential reaction to compression andshearing forces on the transplanted material and the surroundingmaterial.

EXAMPLE 8

A four month old mixed Yorkshire breed pig was subjected to generalanesthesia and placed on its back. The pig was washed and draped in asurgical suite at Harrington Arthritis Research Center, Phoenix, Ariz.The entire surgical procedure was performed aseptically. The lefthind-leg and adjacent abdomen and inguinal area was cleaned with iodine.The knee joint was localized, and the patella localized. A medialincision was performed approximately 3 cm from the posterior part of thepatella and the several subcutis, muscle layers and ligaments was cutapproximately in order to get access to the medial femoral condyle.Using a circular cutter a lesion was prepared in the white cartilage onthe medial part of the medial condyle, leaving a 0.5 to 1 cm margin tothe edge of the cartilage covering the posterior-medial part of thecondyle (left condyle, FIG. 6A). The 0.5 to 1 cm defect was placed in acaudal weight bearing part of the medial condyle. The entire surgicalprocedure was done without tourniquet on the left femur. The differentlayers and skin was sutured appropriately.

On day 3 the animal was again brought to the surgical suite andpositioned as above on the operating table and given general anesthesia.The left hind leg, abdomen and inguinal region was cleaned with iodine,as described above. Sutures were cut and the area opened. It was noticedthat a moderate hematoma was present in the knee joint. The blood clotwas removed and the defect inspected. There was a blood clot in thedefect which was removed. A sterile surgical instrument designed with amale thread cutting edge, with a size corresponding to, or slightlybigger than the circumference of the lesion was carefully screwed downinto the defect. A BioGide® pad was cut to a size equal to the bottom ofthe defect. The first glue used, called Adhesive Protein (A-2707, SigmaChemical, USA) was applied on the dense side of the trimmed hemostaticbarrier pad, and the pad was placed dense side down into the bottom ofthe lesion, using it as a barrier as described above. It was found thatthis glue did not seem to dry very fast. The slight bleeding from thebottom of the defect stopped immediately. A second BioGide® was cutsomewhat bigger in circumference than the lesion and was placed withdense side up (thus the porous side down towards the graft) as describedabove.

This non-cellular covering-pad was then sutured over the cavity, leavingone edge open, where the chondrocyte to be explanted could be injected.The surrounding part of the edge of the pad was covered with the secondglue, Dow Coming Medical Adhesive B (Cat. #895-3, Dow Corning, USA).This second glue dried much faster and more efficiently than the firstglue. It was found that during this particular procedure, the first gluehad not dried sufficiently to hold the hemostatic barrier in place whensuturing of the cap was attempted. Thus, the main barrier formed on theproximal surface of the graft site was by the glue itself.

Using a 1 ml syringe and a 16 gauge needle, the chondrocyte cellsuspension (about 0.6 ml) was drawn up into the barrel of the syringe. A23 gauge short needle was switched for the 16 gauge needle, and the cellsuspension was injected under the sutured covering-patch into the graftsite (about 10×10⁶ cells ). The open edge of the cap was then gluedprior to removal of the needle, and the needle carefully withdrawn. Noleakage of cells was seen. The wound was sutured and as above, notourniquet was used, no bleeding was observed. The final skin layerswere sutured. No protrusion of the skin occurred after suturing, whichindicates that there was no hematoma. Postoperative recovery wasuneventful.

As expected, the grafted chondrocytes produced cartilage matrixsufficient to repair the defect made in the articular cartilage surfaceof the knee joint of the test pig. FIG. 6A is an MRI image of a pig kneeshowing the cartilage defect created in the knee (left condyle, themedial condyle), and FIG. 6B is an MRI image of the same pig knee threemonths after treatment showing repair of the defect.

EXAMPLE 9

A kit comprising the components useful for practicing the method of theinvention, will allow for the convenient practice of the method of theinvention in a surgical setting. In a preferred embodiment, a kit of theinvention will provide sterile components suitable for easy use in thesurgical environment, and will provide a suitable hemostatic barrier,suitable covering patch, and if needed organic glue. A kit of theinvention may also provide sterile, cell-free matrix material suitablefor supporting autologous chondrocytes that are to be implanted into anarticular joint surface defect. In one embodiment, a kit of theinvention contains a Surgicel® hemostatic barrier and a Bio-Gide®covering patch with suitable coating of Tisseel® organic glue, where theSurgicel® and Bio-Gide® have been treated according to the teachings ofthe invention to increase the time till resorption. In instances whereTisseel® is pre-coated, in one embodiment the Tisseel® is supplementedwith additional aprotinin to increase time till resorption.

In another preferred embodiment, the hemostatic barrier andcovering-patch are both a semi-permeable collagen matrix which istreated to extend the time till resorption of the material. It is alsopossible to provide Tisseel® glue in enhanced form as a separatecomponent to be applied as needed because of the inherent variabilityand unique circumstances every repair/transplantation procedure willencounter.

A further embodiment of the kit will include a surgical instrument asdescribed in Example 7 above.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention shown inthe specific embodiments without departing form the spirit and scope ofthe invention as broadly described.

1. An implantable article for cartilage repair by implantation in ananimal comprising a support matrix, and a mixture of chondrocyte cellsand a bio-compatible adhesive adhered to a surface of said supportmatrix, wherein said support matrix is absorbable by the animal.
 2. Theimplantable article of claim 1, wherein said support matrix is asheet-like member capable of supporting growth of the chondrocyte cellsand of providing physical integrity to the implantable article tofacilitate manipulation thereof.
 3. The implantable article of claim 1,wherein said support matrix comprises polypeptides or proteins.
 4. Theimplantable article of claim 1, wherein said support matrix is collagen.5. The implantable article of claim 4, wherein said collagen is selectedfrom the group consisting essentially of equine, porcine, bovine, ovineand chicken collagen.
 6. The implantable article of claim 4, whereinsaid collagen is collagen Type II.
 7. The implantable article of claim1, wherein said support matrix is solid.
 8. The implantable article ofclaim 1, wherein said support matrix is gel-like.
 9. The implantablearticle of claim 4, wherein said collagen is collagen Type I.
 10. Theimplantable article of claim 1, wherein said implantable article isreversibly deformable.
 11. The implantable article of claim 1, whereinsaid support matrix has a rough side.
 12. The implantable article ofclaim 11, wherein said rough side is porous.
 13. The implantable articleof claim 1, wherein said support matrix has a smooth side.
 14. Theimplantable article of claim 1, wherein said support matrix has a roughand a smooth side.
 15. The implantable article of claim 1, wherein saidchondrocyte cells and said bio-compatible adhesive are in an admixture.16. The implantable article of claim 1, further wherein said supportmatrix has a hemostatic effect.
 17. The implantable article of claim 16,wherein said support matrix is a sheet-like member capable of supportinggrowth of the chondrocyte cells and of providing physical integrity tothe implantable article to facilitate manipulation thereof.
 18. Theimplantable article of claim 16, wherein said support matrix comprisespolypeptides or proteins.
 19. The implantable article of claim 16,wherein said support matrix is collagen.
 20. The implantable article ofclaim 19, wherein said collagen is selected from the group consistingessentially of equine, porcine, bovine, ovine and chicken collagen. 21.The implantable article of claim 16, wherein said support matrix issolid.
 22. The implantable article of claim 16, wherein said supportmatrix is gel-like.
 23. The implantable article of claim 19, whereinsaid collagen is collagen Type I.
 24. The implantable article of claim19, wherein said collagen is collagen Type II.
 25. The implantablearticle of claim 16, wherein said implantable article is reversiblydeformable.
 26. The implantable article of claim 16, wherein saidsupport matrix has a rough side.
 27. The implantable article of claim26, wherein said rough side is porous.
 28. The implantable article ofclaim 16, wherein said support matrix has a smooth side.
 29. Theimplantable article of claim 16, wherein said support matrix has a roughand a smooth side.
 30. The implantable article of claim 16, wherein saidchondrocyte cells and said bio-compatible adhesive are in an admixture.